Multi-mer peptides derived from hepatitis C virus envelope proteins for diagnostic use and vaccination purposes

ABSTRACT

Multimer peptides (e.g. 30- to 45-mer peptides) derived from hepatitis C virus envelope proteins reacting with the majority of anti-HCV antibodies present in patient sera are described. The usage of the latter peptides to diagnose, and to vaccinate against, an infection with hepatitis C virus is also disclosed.

FIELD OF THE INVENTION

The present invention relates to multi-mer peptides derived fromhepatitis C virus envelope proteins which react with the majority ofanti-HCV antibodies present in patient sera. Consequently, the presentinvention relates to the usage of the latter peptides to diagnose, andto vaccinate against, an infection with hepatitis C virus.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is a major health problem in bothdeveloped and developing countries. It is estimated that about 1 to 5%of the world population is affected by the virus, amounting up to 175million chronic infections worldwide. HCV infection appears to be themost important cause of transfusion-associated hepatitis and frequentlyprogresses to chronic liver damage. Moreover, there is evidenceimplicating HCV in induction of hepatocellular carcinoma. Consequently,the demand for reliable diagnostic methods and effective therapeuticagents is high. There is also an urgent need to characterize newepitopes which can be used in the design of effective vaccines againsthepatitis C.

HCV is a positive stranded RNA virus of about 9.8 kilobases which codefor at least three structural and at least six non-structural proteins.The structural proteins have not yet been functionally assigned, but arethought to consist of a single core protein and two envelope proteins E1and E2. The E1 protein consists of 192 amino acids and contains 5 to 6N-glycosylation sites, depending on the HCV genotype, whereas the E2protein consists of 363 to 370 amino acids and contains up to 11N-glycosylation sites, depending on the HCV genotype (for review seeMaertens and Stuyver, 1997).

The E1 and E2 proteins are currently not included in HCV antibody (Ab)assays, primarily because of their complex conformational structureswhich require expression in mammalian cells as well as non-denaturingpurification techniques. Indeed, after expression of E2 in Escherichiacoli, the reactivity of HCV sera with the recombinant protein rangedfrom 14 (Yokosuka et al., 1992) to 17% (Mita et al., 1992), whereasexpression in eukaryotic systems yields reactivities of 13 to 97%(Inoue, 1992; Chien, 1993). Others demonstrated that the E1 proteinexpressed as a single protein from eukaryotic cells did not shown highreactivity with patient sera (from 6 to 60%; Kohara et al. (1992), Hsuet al. (1992), Chien et al. (1993)). We previously reported that highprevalences of Ab's to both of the purified recombinant E1 and E2proteins, which were expressed in mammalian cells, could be found insera from chronic hepatitis C patients (WO 96/04385 to Maertens et al.).In this regard, we also demonstrated that the majority of anti-E1 andanti-E2 antibodies in sera from HCV patients could not be mapped using20-mer peptides (WO 96/04385 to Maertens et al.). Indeed, although allof the murine monoclonal Ab's against E1 could be mapped to reactivitywith two 20-mer peptides, denoted as epitope A (amino acids (aa)313-326) and epitope B (aa 208-224), at most 50% of patient serareactive with recombinant proteins recognized epitope A and B. Withregard to the E2 protein, only three out of twenty four murinemonoclonal Ab's could be mapped using 20-mer peptides. These three Ab'swere mapped to the hypervariable region I (HVR I) covered by peptideE2-67 (aa 394-413) and to a region covered by a peptide denoted E2-13B(aa 523-542). The remaining twenty-one Ab's could not be mapped using20-mer peptides. The relative map positions of seven of these Ab's couldbe deduced from competition studies using recombinant E2 protein.

Taken together, it appears that anti-E1 and anti-E2 Ab's might be highlyprevalent in sera of HCV patients. However, determining the presence ofthese Ab's is problematic due to the need to use eukaryoticallyexpressed E1 and E2, which have to be purified using cumbersomenon-denaturing techniques. As an alternative, chemically synthesized20-mer peptides derived from the E1 and/or E2 proteins were produced.However, these synthesized 20-mer peptides were not able to recognizethe anti-E1 and anti-E2 Ab's in sera from HCV patients.

There is thus a need to design alternative methods to screen for HCVenvelope Ab's.

AIMS OF THE INVENTION

It is clear from the literature cited above that the E1 and E2 proteinsprobably have complex conformational structures which are essential forrecognizing (and binding to) the anti-E1 and anti-E2 Ab's in sera fromHCV patients. This could explain why prokaryotically expressed completeor near-complete E1 and E2 proteins, which might be malfolded due to thelack of glycosylations, relevant chaperones or correct cysteine bridges,and 20-mer peptides, which might be unable to mimic a complexconformational structure, are not able to recognize these Ab's.

The present invention relates to the surprising finding that multi-merpeptides (eg 30- to 45-mer peptides) are able to recognize the majorityof anti-E1 and anti-E2 Ab's in sera from HCV patients. It should beclear that this is a surprising finding because there is no guidancewhich would suggest that 30- to 45-mer peptides derived from E1 and E2would acquire proper folding and would efficiently recognize themajority of HCV envelope Ab's. In contrast, one would assume that thechance that multi-mer peptides malfold would be as great, or evengreater, than the chance that prokaryotically expressed completeproteins malfold as is suggested above. In the case of the HCV NS3protein for example, which reacts with more than 90% of patient samplesas expressed from E. coli, 20-50 mer peptides only react very weakly.

Therefore, the present invention aims at providing a peptide of morethan 20 contiguous amino acids derived from the envelope region ofHCV-related viruses which binds and recognizes anti-HCV-related virusantibodies. HCV-related viruses, including HCV, GBV-B virus, GBV-A virusand GBV-C (HGV or hepatitis G virus), are a division of theFlaviviruses, which further comprise Dengue virus, Yellow fever virus,Pestiviruses such as Classical Swine Fever Virus and Bovine ViralDiarrhea Virus (Wengler, 1991).

More specifically, the present invention aims at providing a peptidewhich binds and recognizes an anti-HCV antibody or an anti-HGV antibodypresent in a sample of body fluid and which is chosen from the groupconsisting of the sequences as represented in SEQ ID NOs 1 to 38 (seeTable 1) or a functionally equivalent variant or fragment thereof.

In this respect, the present invention aims specifically at providing apeptide as described above, wherein said anti-HCV antibody present in asample of body fluid is an anti-HCV-E1 antibody or an anti-HCV-E2antibody.

The present invention thus aims also at providing a peptide as describedabove, wherein said anti-HGV antibody present in a sample of body fluidis an anti-HGV-E1 antibody or an anti-HGV-E2 antibody.

Moreover, the present invention aims at providing a peptide as describedabove, wherein said peptide is synthesized chemically or is synthesizedusing recombinant DNA techniques.

The present invention also aims at providing a peptide as describedabove, wherein said peptide is biotinylated or contains cysteinebridges.

Furthermore, the present invention aims at providing any combination ofpeptides as described above, as well as compositions containing saidcombination of peptides or peptides as described above.

In addition, the present invention aims at providing a method fordiagnosing exposure to or infection by HCV-related viruses comprisingcontacting anti-HCV-related virus antibodies within a sample of bodyfluid with a peptide as described above or with a combination ofpeptides as described above, determining the binding of anti-HCV-relatedvirus antibodies within a sample of body fluid with a peptide asdescribed above or with a combination of peptides as described above.

In addition, the present invention aims at providing an assay kit fordetecting the presence of anti-HCV-related virus antibodies within asample of body fluid comprising a solid support, a peptide as describedabove or a combination of peptides as described above, appropriatemarkers which allow to determine the complexes formed betweenanti-HCV-related virus antibodies within a sample of body fluid with apeptide as described above or a combination of peptides as describedabove.

In addition, the present invention aims at providing a bioassay foridentifying compounds which modulate the interaction between a peptideand an anti-HCV-related virus antibody, said bioassay comprisingcontacting anti-HCV-related virus antibodies with a peptide as describedabove or a combination of peptides as described above, determining thebinding of anti-HCV-related virus antibodies with a peptide as describedabove or a combination of peptides as described above, adding amodulator (ie a compound which is able to modulate the interactionbetween an envelope protein and an anti-HCV-related virus antibody) or acombination of modulators to the contacted anti-HCV-related virusantibodies with a peptide as described above or a combination ofpeptides as described above, determining the modulation of binding ofanti-HCV-related virus antibodies with a peptide as described above or acombination of peptides as described above

In addition, the present invention aims at providing a bioassay foridentifying compounds which modulate the interaction between a peptideand an anti-HCV-related virus antibody, said bioassay comprisingdetermining the binding of anti-HCV-related virus antibodies with apeptide as described above or a combination of peptides as describedabove, contacting a modulator with a peptide as described above or acombination of peptides as described above, adding anti-HCV-relatedvirus antibodies to the contacted modulator with the peptide asdescribed above or a combination of peptides as described above,determining the modulation of binding between anti-HCV-related virusantibodies with a peptide as described above or a combination ofpeptides as described above.

Moreover, the present invention aims at providing a modulator, acomposition containing a modulator, or a combination of modulators whenproduced by the bioassay as described above or when identified by theabove-described bioassays.

Moreover, the present invention aims at providing a compositioncomprising a plasmid vector comprising a nucleotide sequence encoding apeptide as described above, or a modulator as described above, operablylinked to transcription regulatory elements.

Moreover, the present invention aims at providing a composition asdescribed above for use to vaccinate or therapeutically treat humansagainst infection with HCV-related virus or any mutated strain thereof.

Moreover, it is an aim of the present invention to provide an antibody,more particularly a monoclonal antibody, characterized in that itspecifically recognizes an HCV-related virus polypeptide as describedabove.

Finally, it is an aim of the present invention to provide a method toimmunize humans against infection with HCV-related virus or any mutatedstrain thereof, comprising the use of a peptide as described above or acombination of peptides as described above.

All the aims of the present invention are considered to have been met bythe embodiments as set out below. Other advantages and features of theinstant invention will be evident from the following claims and detaileddescription.

BRIEF DESCRIPTION OF TABLES AND DRAWINGS

Table 1 provides information on the envelope protein and the HCVgenotype from which the peptides of the present invention are derived.This table also provides the name, the amino acid sequence, the positionwithin the envelope proteins and the sequence identity (SEQ ID) of thepeptides of the present invention.

Table 2 shows ELISA results (in mOD) of reactivities of multimerpeptides and recombinant E2 with 60 HCV positive samples and 4 controlsamples.

Table 3 shows the analysis for E1 antibodies of 23 sera from respondersto interferon treatment.

Table 4 shows the analysis of E2 antibodies of 23 sera from respondersto interferon treatment.

Table 5 shows the monitoring of disease over time by measuringantibodies to the HCV E1 and E2 regions in 18 patients.

Table 6 indicates the reactivity of HGV (Hepatitis G virus) RNA positivesera with the HGV E1 peptide V1V2.

FIG. 1 demonstrates the positions of the multi-mer peptides of thepresent invention relative to the conserved and variable regions of theE1 envelope protein of HCV (HVR=hypervariable regions; V=variableregions; C=conserved regions; HR=hydrophobic region; SA=signal anchordomain; Y=glycosylation; I=cysteine).

FIG. 2 demonstrates the positions of the multi-mer peptides of thepresent invention relative to the conserved and variable regions of theE2 envelope protein of HCV (HVR=hypervariable regions; V=variableregions; C=conserved regions; SA=signal anchor domain; Y=glycosylation;I=cysteine).

FIG. 3 shows the reactivity of 20-mer E2 peptides. The OD values ofserum samples from patients with chronic active hepatitis C were addedand plotted against the different peptides.

FIG. 4 shows the reactivity of mulit-mer E2 peptides. The OD values ofthe samples were added and plotted against the different peptides. Thesamples were identical as used for FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein draws on previously published work andpending patent applications. By way of example, such work consists ofscientific papers, patents or pending patent applications. All thesepublications and applications, cited previously or below are herebyincorporated by reference.

The present invention is based on the finding that multimer peptides, asof a certain length, derived from the envelope proteins of HCV-relatedviruses, eg HCV and HGV, recognize and bind anti-HCV-related virusantibodies, eg anti-HCV antibodies and anti-HGV antibodies,respectively. Therefore, the present invention provides a peptide ofmore than 20 contiguous amino acids derived from the envelope region ofHCV-related viruses which binds and recognizes anti-HCV-related virusantibodies.

HCV-related viruses include, but are not limited to HCV, GBV-B virus,GBV-A virus and GBV-C virus (HGV or hepatitis G virus)(Linnen et al.,1996). HCV constitutes a genus within the Flaviviridae, and is closelyrelated to hepatitis G virus (26.8% at the amino acid level). The term“envelope region” of HCV-related viruses is a well-known region by aperson skilled in the art (Wengler, 1991), and comprises the E1 proteinas well as the E2 protein, which was previously called non-structuralprotein 1 (NS1) or E2/NS1.

Furthermore, the present invention relates to a peptide, which binds andrecognizes an anti-HCV antibody or an anti-HGV antibody present in asample of body fluid, and which is chosen from the group consisting ofthe sequences as represented in SEQ ID 1 to 38 (see Table 1) or afunctionally equivalent variant or fragment thereof.

The present invention relates also to a peptide as described above,wherein said anti-HCV antibody or said anti-HGV antibody present in asample of body fluid is an anti-HCV-E1 or anti-HCV-E2 antibody, or ananti-HGV-E1 or anti-HGV-E2 antibody, respectively.

The term “a peptide” refers to a polymer of amino acids (aa's) derived(i.e. containing less aa's) from the well-known HCV-related virusenvelope proteins E1 and E2 (Linnen et al., 1996, Maertens and Stuyver,1997), which binds anti-HCV-related virus antibodies. The term “apeptide” refers in particular to a polymer of aa's derived from HCVenvelope proteins E1 and E2, which binds anti-HCV antibodies, or fromHGV envelope proteins E1 and E2, which binds anti-HGV antibodies.

The terms “peptide”, “polypeptide” and “protein” are usedinterchangeably herein.

The term “an anti-HCV-related virus antibody” refers to any polyclonalor monoclonal antibody binding to a HCV-related virus particle or anymolecule derived from said viral particle. More particularly, the term“an anti-HCV-related virus antibody” refers to antibodies binding to thenatural, recombinant or synthetic E1 and/or E2 proteins derived from HCVor HGV proteins (anti-HCV-E1 or anti-HCV-E2 antibody, or anti-HGV-E1 oranti-HGV-E2 antibody, respectively).

The term “monoclonal antibody” used herein refers to an antibodycomposition having a homogeneous antibody population. The term is notlimiting regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made.

In addition, the term “antibody” also refers to humanized antibodies inwhich at least a portion of the framework regions of an immunoglobulinare derived from human immunoglobulin sequences and single chainantibodies as described in U.S. Pat. No. 4,946,778 and to fragments ofantibodies such as F_(ab), F_(′(ab)2), F_(v), and other fragments whichretain the antigen binding function and specificity of the parentantibody.

The term “a sample of body fluid” as used herein refers to a fluidobtained from an organism, such as serum, plasma, saliva, gastricsecretions, mucus, spinal cord fluid, and the like.

The term “the group consisting of the sequences as represented in SEQ IDNOs 1 to 38” as used herein refers to the thirty-eight peptides shown inTable 1 of the present application. In this table, it is indicated:

-   -   in the column named “protein” from which HCV envelope protein        the peptide is derived, but for the envelope protein of HGV,        which is denoted E1 (HGV),    -   in the column named “genotype” the HCV genotype from which the        envelope protein is derived, and thus the peptide is derived,        except for HGV which was not determined (ND),    -   in the column named “peptide” the assignment of the peptide        region.    -   the aa sequence of the peptide and,    -   in the column named “position”, the well-known (Maertens and        Stuyver, 1997) aa position of the peptides within the HCV        envelope proteins. Note that the position for the E1 envelope        protein is not determined, which is denoted as “ND”.

The term “functionally equivalent” as used in “functionally equivalentvariant or fragment thereof” refers to variants and fragments of thepeptides represented by SEQ ID 1 to 38, which bind anti-HCV-relatedvirus antibodies. The term “variant or fragment” as used in“functionally equivalent variant or fragment thereof” refers to anyvariant or any fragment of the peptides represented by SEQ ID 1 to 38.Furthermore, the latter terms do not refer to, nor do they exclude,post-translational modifications of the peptides represented by SEQ ID 1to 38 such as glycosylation, acetylation, phosphorylation, modificationswith fatty acids and the like. Included within the definition are, forexample, peptides containing one or more analogues of an aa (includingunnatural aa's), peptides with substituted linkages, mutated versions ornatural sequence variations of the peptides (for example correspondingto the genotypes HCV, as described in WO 94/12670 to Maertens et al.),peptides containing disulfide bounds between cysteine residues, or othercysteine modifications, biotinylated peptides, as well as othermodifications known in the art. Modification of the structure of thepolypeptides can be for such objectives as increasing therapeutic orprophylactic efficacy, stability (e.g. ex vivo shelf life and in vivoresistance to proteolytic degradation), or post-translationalmodifications (e.g. to alter the phosphorylation pattern of protein).Such modified peptides, when designed to retain at least one activity ofthe naturally-occurring form of the protein are considered functionalequivalents of the polypeptides described in more detail herein. Suchmodified peptides can be produced, for instance, by amino acidsubstitution, deletion, or addition. For example, it is reasonable toexpect that an isolated replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar replacement of an amino acid with a structurally related aminoacid (i.e. isosteric and/or isoelectric mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids can be divided into four families: (1) acidic: aspartate,glutamate; (2) basic: lysine, arginine, histidine; (3) nonpolar:alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar: glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. In similar fashion,the amino acid repertoire can be grouped as (1) acidic: aspartate,glutamate; (2) basic: lysin, arginine histidine, (3) aliphatic: glycine,alanine, valine, leucine, isoleucine, serine, threonine, with serine andthreonine optionally be grouped separately as aliphatic-hydroxyl; (4)aromatic: phenylalanine, tyrosine, tryptophan; (5) amide: asparagine,glutamine; and (6) sulfur-containing: cysteine and methionine (see, forexample, Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.:1981). Whether a change in the amino acid sequence of a peptide resultsin a functional homologue (e.g. functional in the sense that theresulting polypeptide mimics the wild-type form) can be readilydetermined by assessing the ability of the variant peptide to produce aresponse in e.g. ELISAs in a fashion similar to the wild-type protein,or to competitively inhibit such a response. Polypeptides in which morethan one replacement has been introduced can be readily tested in thesame manner.

It should also be clear that the region of a peptide represented by SEQID 1 to 38 which bind to an antibody (the so-called epitope) need not tobe composed of a contiguous aa sequence.

In this regard, the term “fragment” includes any fragment whichcomprises these non-contiguous binding regions or parts thereof. Inother words, fragments which include these binding regions may beseparated by a linker, which is not a functional part of the epitope.This linker does not need to be an amino acid sequence, but can be anymolecule, eg organic or inorganic, that allows the formation of thedesired epitope by two or more fragments.

Moreover, it should be clear that the variants and fragments of SEQ IDNOs 1 to 5, 7 to 9, and 18 as used herein include peptides having alength of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31aa's, or 32 aa's, or 33 aa's, or 34 aa's. Moreover, it should be clearthat the variants and fragments of SEQ ID NO 6 as used herein include topeptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's.Moreover, it should be clear that the variants and fragments of SEQ IDNO 10 as used herein include to peptides having a length of at least 21aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27aa's, or 28 aa's. Moreover, it should be clear that the variants andfragments of SEQ ID NOs 11, 15, 21, 34 as used herein include topeptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or30 aa's, or 31 aa's, or 32 aa's, or 33 aa's. Moreover, it should beclear that the variants and fragments of SEQ ID NOs 12, 24 or 32 as usedherein include to peptides having a length of at least 21 aa's, or 22aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34aa's, or 35 aa's. Moreover, it should be clear that the variants andfragments of SEQ ID NOs 13, 22, or 34 used herein include to peptideshaving a length of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's,or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's.Moreover, it should be that clear the variants and fragments of SEQ IDNO 16 as used herein include to peptides having a length of at least 21aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39aa's, or 40 aa's. Moreover, it should be clear that the variants andfragments of SEQ ID NO 17 as used herein refers to peptides having alength of at least 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31aa's. Moreover, it should be clear that the variants and fragments ofSEQ ID NO 19 as used herein refers to peptides having a length of atleast 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's. Moreover,it should be clear that the variants and fragments of SEQ ID NOs 20 and30 as used herein include to peptides having a length of at least 21aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27aa's. Moreover, it should be clear that the variants and fragments ofSEQ ID NO 23 as used herein include to peptides having a length of atleast 21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26aa's, or 27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38aa's, or 39 aa's, or 40 aa's, or 41 aa's, or 42 aa's; or 43 aa's.Moreover, it should be clear that the variants and fragments of SEQ IDNOs 25 or 29 as used herein include peptides having a length of at least21 aa's, or 22 aa's, or 23 aa's, or 24 aa's. Moreover, it should beclear that the variants and fragments of SEQ ID NO 26 as used hereininclude peptides having a length of at least 21 aa's, or 22 aa's, or 23aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29aa's. Moreover, it should be clear that the variants and fragments ofSEQ ID NO 27 as used herein include peptides having a length of at least21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or33 aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39aa's, or 40 aa's, or 41 aa's, or 42 aa's, or 43 aa's, or 44 aa's.Moreover, it should be clear that the variants and fragments of SEQ IDNO 28 or 31 as used herein include peptides having a length of at least21 aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or27 aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's.Moreover, it should be clear that the variants and fragments of SEQ IDNO 33 as used herein include peptides having a length of at least 21aa's, or 22 aa's, or 23 aa's, or 24 aa's, or 25 aa's, or 26 aa's, or 27aa's, or 28 aa's, or 29 aa's, or 30 aa's, or 31 aa's, or 32 aa's, or 33aa's, or 34 aa's, 35 aa's, or 36 aa's, or 37 aa's, or 38 aa's, or 39aa's, or 40 aa's, or 41 aa's. Moreover, it should be clear that thevariants and fragments of SEQ ID NOs 14 or 37 as used herein includepeptides having a length of at least 21 aa's, or 22 aa's, or 23 aa's, or24 aa's, or 25 aa's, or 26 aa's, or 27 aa's, or 28 aa's, or 29 aa's, or30 aa's, or 31 aa's, or 32 aa's, or 33 aa's, or 34 aa's, 35 aa's, or 36aa's, or 37 aa's, or 38 aa's, or 39 aa's.

In addition, it shall be appreciated by the person skilled in the artthat the amino acid regions of the peptides, which are disclosed in thepresent invention and that bind anti-HCV antibodies, can be delineatedin more detail by experimentation.

In addition, it should be clear that the variants and fragments of thepeptides represented by SEQ ID 1 to 38, as herein described, can beprepared by any method known in the art such as classical chemicalsynthesis, as described by Houbenweyl (1974) and Atherton & Shepard(1989), or by means of recombinant DNA techniques as described by egManiatis et al. (1982), or Sambrook et al. (1989).

Similarly, it should be clear that also the peptides represented by SEQID 1 to 38 of the present invention can be prepared by any method knownin the art and more particularly by means of classical chemicalsynthesis, as described by Houbenweyl (1974) and Atherton & Shepard(1989), or by means of recombinant DNA techniques such as described byeg Maniatis et al. (1982), or Sambrook et al. (1989).

The present invention further relates to the peptides represented by SEQID 1 to 38 and functionally equivalent variants or fragments thereof,all as defined above, which are biotinylated or contain cysteinebridges. Biotinylated peptides can be obtained by any method known inthe art, such as the one described in WO93/18054 to De Leys. Methods forobtaining peptides containing inter- and/or intramolecular cysteinebridges are extensively described in WO 96/13590 to Maertens & Stuyver.

The present invention also relates to any combination of peptidesrepresented by SEQ ID 1 to 38 and functionally equivalent variants orfragments thereof as defined above. The terms “any combination” refersto any possible mixture of above-described peptides or any possiblelinkage (covalently or otherwise) between above-described peptides.Examples of the latter peptide combinations are simple mixtures, homo-or hetero-branched peptides, combinations of biotinylated peptidespresented on streptavidin, avidin or neutravidin, chemicallycross-linked peptides with or without spacer, condensed peptides andrecombinantly produced peptides.

The present invention relates also an antibody, more particularly amonoclonal antibody, characterized in that it specifically recognizes anHCV-related virus polypeptide as described above.

The present invention also relates to a method for diagnosing exposureto or infection by HCV-related viruses comprising contactinganti-HCV-related virus antibodies within a sample of body fluid with apeptide as described above or with a combination of peptides asdescribed above, and, determining the binding of anti-HCV-related virusantibodies within a sample of body fluid with a peptide as describedabove or with a combination of peptides as described above.

As used herein, the term “a method for diagnosing” refers to anyimmunoassay known in the art such as assays which utilize biotin andavidin or streptavidin, ELISAs and immunoprecipitation and agglutinationassays. A detailed description of these assays is given in WO 96/13590to Maertens & Stuyver.

In this regard, the present invention also relates to an assay kit fordetecting the presence of anti-HCV-related virus antibodies comprising asolid support, a peptide as described above or a functionally equivalentvariant or fragment thereof, or combination of peptides as describedabove, and appropriate markers which allow to determine the complexesformed between anti-HCV-related virus antibodies within a sample of bodyfluid with a peptide as described above, or a functionally equivalentvariant or fragment thereof, or combination of peptides as describedabove.

The term “a solid support” refers to any solid support known in the art.

Similarly, the term “appropriate markers” refers to any marker known inthe art.

It should also be clear that the term “a method for diagnosing”encompasses screening, detection, confirmation, monitoring andserotyping methods.

The present invention further pertains to a bioassay for identifyingcompounds which modulate the binding between a peptide and ananti-HCV-related virus antibody, comprising contacting anti-HCV-relatedvirus antibodies with a peptide as described above, or a combination ofpeptides as described above, and determining the binding ofanti-HCV-related virus antibodies with a peptide as described above, ora combination of peptides as described above, adding a modulator or acombination of modulators to the contacted anti-HCV-related virusantibodies with a peptide as described above, or a combination ofpeptides as described above, and finally determining the modulation ofbinding of anti-HCV-related virus antibodies with a peptide as describedabove, or a combination of peptides as described above.

In another embodiment the present invention features a bioassay foridentifying compounds which modulate the binding between a peptide andan anti-HCV-related virus antibody, comprising determining the bindingof anti-HCV-related virus antibodies with a peptide as described above,or a combination of peptides as described above, contacting a modulatorwith a peptide as described above, or a combination of peptides asdescribed above, adding anti-HCV-related virus antibodies to thecontacted modulator with a peptide as described above, or a combinationof peptides as described above, determining the modulation of binding ofanti-HCV-related virus antibodies with a peptide as described above, ora combination of peptides as described above.

The term “compound” as used herein refers to a composition, which has amolecular weight of less than about 25 KDa, preferably less than 10 KDa,and most preferably less than 5 KDa. Compounds can be nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules, or antibodies which may be generated bythe host itself upon vaccination.

The term “binding” as used herein indicates that a peptide as describedabove is physically connected to, and interacts with antibodies. Bindingof the peptide to the antibody can be demonstrated by any method orassay known in the art such as binding-, ELISA, and RIA-type of assaysor competition assays (ea see Examples section and Current protocols inimmunology).

The terms “modulation” or “modulate” as used herein refer to bothupregulation (i.e., activation or stimulation (e.g., by agonizing orpotentiating)) and downregulation (i.e. inhibition or suppression (e.g.by antagonizing, decreasing or inhibiting) of the binding between apeptide and an anti-HCV antibody.

The term “modulator” as used herein refer to the ability of a compoundas described above to modulate as described above.

The term “peptidomimetics” as used herein refers to molecules which canbe manufactured and which mimic those residues of peptides whichmodulate the interaction of the antibody with the peptide as describedabove. For instance, non-hydrolyzable peptide analogs of such residuescan be generated using benzodiazepine (e.g., see Freidinger et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), PNA, substituted gamma lactam rings (Garveyet al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988), ketomethylene pseudopeptides(Ewenson et al. (1986) J Med Chem 29:295: and Ewenson et al. inPeptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagrai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun, 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71).

The present invention pertains to a modulator produced by a bioassay asdescribed above.

The present invention pertains also to a modulator for the interactionbetween a peptide and an anti-HCV-related virus antibody, when saidmodulators are identified by a bioassay as described above.

The present invention features a composition comprising as an activesubstance a peptide as described above or a combination of peptides asdescribed above.

The present invention features also a composition comprising as anactive substance a modulator as described above or a combination ofmodulators as described above.

In another embodiment, the present invention relates to a compositioncomprising a plasmid vector comprising a nucleotide sequence encoding apeptide as described above, operably linked to transcription regulatoryelements. Upon introduction in a human tissue said plasmid vectorinduces the expression in vivo, of the nucleotide sequence therebyproducing the encoded protein. If said protein elicits an immunogenicresponse it is referred to as a DNA vaccine. It is readily apparent tothose skilled in the art that variations or derivatives of thenucleotide sequence can be produced which alter the nucleotide sequence.The altered polynucleotide may have an altered nucleic sequence, yetstill encodes a protein as described above, and which reacts withanti-HCV-related virus antibodies, and is considered a to be functionalequivalent.

In a preferred embodiment, the present invention relates to acomposition as described herein for use as to vaccinate humans againstinfection with HCV-related virus or any mutated strain thereof.

In another preferred embodiment, the present invention relates to acomposition as described herein for use as to therapeutically treathumans against infection with HCV-related virus or any mutated strainthereof.

A composition of the present invention can be, as appropiate, any of thepreparations described herein, including peptides, functionallyequivalent variants or fragments thereof, a combination of peptides, ormodulators (e.g. as identified in the bioassay provided herein).Specifically, the term “a composition” relates to an immunogeniccomposition capable of eliciting protection against HCV-related virus,in particular against HCV and/or HGV, whether partial or complete. Theterm “as an active substance” relates to the component of the vaccinecomposition which elicits protection against HCV-related viruses, inparticular against HCV and/or HGV. An active substance (e.g. thepeptides or the modulators of the present invention) can be used assuch, in a biotinylated form (as explained in WO 93/18054) and/orcomplexed to Neutralite Avidin according to the manufacturer'sinstruction sheet (Molecular Probes Inc., Eugene, Oreg.).

It should also be noted that “a composition” comprises, in addition toan active substance, a suitable excipient, diluent, carrier and/oradjuvant which, by themselves, do not induce the production ofantibodies harmful to the individual receiving the composition nor dothey elicit protection. Suitable carriers are typically large slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric aa's, aa copolymers and inactivevirus particles. Such carriers are well known to those skilled in theart. Preferred adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: aluminium hydroxide, aluminium incombination with 3-O-deacylated monophosphoryl lipid A as described inWO 93/19780, aluminium phosphate as described in WO 93/24143,N-acetyl-muramyl-L-threonyl-D-isoglutamine as described in U.S. Pat. No.4,606,918, N-acetyl-normuramyl-Lalanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine2(1′2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine and RIBI (ImmunoChem Research Inc., Hamilton, Mont.), whichmay contain one or all of the following elements: monophosphoryl lipid A(detoxified endotoxin), trehalose-6,6-dimycolate, and cell wall skeleton(MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the threecomponents MPL, TDM or CWS may also be used alone or combined 2 by 2.Additionally, adjuvants such as Stimulon (Cambridge Bioscience,Worcester, Mass.), MF 57 (Chiron) or SAF-1 (Syntex) may be used, as wellas adjuvants such as combinations between QS21 and 3-de-O-acetylatedmonophosphoryl lipid A (WO94/00153), or MF-59 (Chiron), orpoly[di(carboxylatophenoxy)phosphazene] based adjuvants (Virus ResearchInstitute), or blockcopolymer based adjuvants such as Optivax (Vaxcel)or GammaInulin (Anutech), or Gerbu (Gerbu Biotechnik). Furthermore,Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA)may be used for non-human applications and research purposes. “Acomposition” will further contain excipients and diluents, which areinherently non-toxic and non-therapeutic, such as water, saline,glycerol, ethanol, wetting or emulsifying agents, pH bufferingsubstances, preservatives, and the like. Typically, a vaccinecomposition is prepared as an injectable, either as a liquid solution orsuspension. Solid forms, suitable for solution on, or suspension in,liquid vehicles prior to injection may also be prepared. The preparationmay also be emulsified or encapsulated in liposomes for enhancingadjuvant effect. The polypeptides may also be incorporated into ImmuneStimulating Complexes together with saponins, for example Quil A(ISCOMS). Compositions, which can be used as a vaccine, comprise animmunologically effective amount of the polypeptides of the presentinvention and/or modulators, as well as any other of the above-mentionedcomponents. “Immunologically effective amount” means that theadministration of that amount to an individual, either in a single dosisor as part of a series, is effective for prevention or treatment. Thisamount varies depending upon the health and physical condition of theindividual to be treated, the taxonomic group of the individual to betreated (e.g. nonhuman primate, primate, etc.), the capacity of theindividual's immune system to mount an effective immune response, thedegree of protection desired, the formulation of the vaccine, thetreating's doctor assessment, the strain of the infecting HCV and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.Usually, the amount will vary from 0.01 to 1000 μg/dose, moreparticularly from 0.1 to 100 μg/dose. Compositions, which can be used asa vaccine are conventionally administered parenterally, typically byinjection, for example, subcutaneously or intramuscularly.

In the case of DNA vaccines, particular useful methods for eliciting animmune response include the coating of gold particles with the plasmidvector encoding the desired peptide, and injecting them under highpressure into the epidermis and/or dermis, eg by means of a devicecalled gene gun (eg as produced by Powderject Vaccines, Madison, Wis.,USA).

Additional formulations suitable for other methods of administrationinclude oral formulations and suppositories. Dosage treatment may be asingle dose schedule or a multiple dose schedule. The vaccine may beadministered in conjunction with other immunoregulatory agents. Itshould be noted that a vaccine may also be useful for treatment of anindividual, in which case it is used as a to “therapeutically treathumans”.

As used herein, a “plasmid vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. Preferred vectors are those capable of autonomous replicationand/or expression of nucleic acids to which they have been linked. Ingeneral, but not limited to those, plasmid vectors are circular doublestranded DNA loops which, in their vector form, are not bound to thechromosome. For expression purposes, promoters are required. For DNAvaccination, a very suitable promoter is the Major Immediate Early (MIE)of human cytomegalovirus.

As used herein, a “nucleotide sequence” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and single(sense or antisense) and double-stranded polynucleotides.

As used herein, the term “transcription regulatory elements” refers to anucleotide sequence which contains essential regulatory elements, iesuch that upon introduction into a living vertebrate cell it is able todirect the cellular machinery to produce translation products encoded bythe polynucleotide.

The term “operably linked” refers to a juxtaposition wherein thecomponents are configured so as to perform their usual function. Thus,transcription regulatory elements operably linked to a nucleotidesequence are capable of effecting the expression of said nucleotidesequence. Those skilled in the art can appreciate that differenttranscriptional promoters, terminators, carrier vectors or specific genesequences may be used succesfully.

Finally, the present invention provides a method to immunize humansagainst infection with HCV-related virus or any mutated strain thereof,comprising the use of a peptide as described above or a combination ofpeptides as described above.

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and can not be construed as to restrict the invention inany way.

EXAMPLES CL Example 1 Synthesis of Multimer E1 and E2 Peptides

We aimed at synthesizing peptides which would display epitopes, similarto the ones present on E1 and E2 peptides expressed in mammalian cells.Since such epitopes do not seem to be present in E1 and E2 proteinsexpressed in E. coli, the design of such peptides was not an easy task.We first aligned E1 and E2 primary amino acid sequences of different HCVgenotypes and delineated variable and constant domains. It was reasonedthat these domains, or a combination of two or more of these domainsmight represent conformational domains, ie form or constituteindependent conformational units. If displayed as 3D structure, theseconformational domains may also contain conformational epitopes. Thelatter domains may therefore be able to adopt a native-like structure asis present in the envelope proteins when these envelope proteins areexpressed in mammalian cells. In contrast such structures are absentwhen the envelope proteins are expressed in prokaryotic cells, like E.coli.

The following domains were assigned:

V1, V2, V3, V4, V5, V6=variable regions; C1, C2, C3, C4=conserveddomains; HR=hydrophobic region; SA=signal anchor sequence; HVRI,HVRII=hypervariable regions of E2. Protein Region Amino acid position E1V1 192-203 C1 204-217 V2 218-223 C2 224-229 V3 230-242 C3 243-247 V4248-257 HR 258-293 V5 294-303 C4 304-329 V6 330-342 SA 343-383 E2 HVRI384-411 C1 412-470 HVRII 471-482 C2 483-521 V3 522-548 C3 549-569 V4570-580 C4 581-704 SA 705-746

Based on these domains of the BE11 subtype 1b isolate (SEQ ID 50 inPCT/EP 95/03031), we designed a series long peptides of 24 to 45 aminoacids. For some extended domains of the envelope proteins more than onemultimer peptide was synthesized in order to encompass the domain ofinterest. Table 1 gives an overview of the peptides with theirrespective amino acid positions; numbering starts from the firstinitiation codon of the HCV polyprotein. Peptides were synthesized usingt-Boc technology as explained in detail in WO 93/18054.

Example 2 Reactivity of Multimer Peptides with E1 and E2 Antibodies inPatient Sera

A series of 60 randomly chosen samples from patients with chronic activehepatitis C were tested for reactivity with the multimer peptides. Thesesamples did not show any notable reactivity with 20-mer peptides exceptfor some 20-mer peptides derived from the HVRI. For comparison,reactivity with the hydrophylic ectodomain of E2, the recombinant E2hprotein, was assayed (E2h extends from aa 384-708 and was cloned fromSEQ ID NO 45, and expressed and purified as described in PCT/EP95/03031). Peptides were coated onto streptavidin-coated plates (5μg/ml) and antibodies in serum samples were left to react and detectedusing the reagents and procedures as described in the package insert ofthe INNOTEST HCV Ab III kit (Innogenetics, Gent, Belgium). Table 2 showsthe results of the ELISA tests, in which a cutoff of 150 mOD was used.In this series, 5 sera did not show reactivity with the E2h protein,only one of these reacted with the HVRI peptide. Five out of 60 sera(8%; e.g. sample 17758) only reacted with the E2h protein, 34 (57%)recognized HVRI, 24 (40%) reacted with C1-a, 18 (30%) with C1-b, 21(35%) with HVRII, 17 (28%) C2-a, 22 (37%) with C2-b, 18 (30%) with C3,18 (30%) with C3′, 17 (28%) with C3″, 18 (30%) with V4, 22 (37%) withC4, 21 (35%) with C4-a, 35 (58%) with C4-b, and 24 (40%) with C4-c. Thisexperiment surprisingly learned that, while none of the samplesrecognized any of the 20-mer peptides, except for those derived from theHVRI, 50 out of 55 (91%) E2h reactive sera could be detected using thepeptides of the present invention.

In a second series of 23 sera derived from chronic hepatitis C patientswho were long-term responders to interferon-alpha treatment and 3 HCVinfected chimpanzees, E1 and E2 antibodies were tested. Eighteen out of23 samples (78%) reacted with recombinant E1s protein, expressed andpurified from mammalian cells as described in PCT/EP 95/03031. Ninesamples (39%) reacted with the C4V6 region, another 9 (39%) with theV1V2 region, and 3 with V2V3 (Table 4). For comparative purposes peptideV5, ie SQLFTISPRRHETVQD, is shown.

Different reactivities to E2 were observed (Table 4) as compared withthe first series of samples. 21 samples (91%) reacted with E2h, with 13(57%) reactive on HVRI, 9 (39%) with C1-a, 11 (48%) with C1-b, 1 withHVRII, C2-a, and C2-b each, 2 with C3, 3 with C4-a, 4 (17%) with C4-b,and 4 (17%) with C4-c. In this series of patients with a benignevolution of disease, the C1 region was more frequently recognized andfewer antibodies to the C4 region were detected as compared to theseries of samples obtained from patients with chronic active hepatitis.These results indicate that peptides from the C1, C2, and C4 regions maybe particularly useful in monitoring the course on HCV-related virusdisease. More specifically, antibodies to the C1 region may betterneutralize HCV as compared to anti-C4 antibodies. The C1 domain maytherefore be functionally important, eg exhibit receptor-bindingactivity. Neutralization of this region may therefore result in lesseractivity of the disease and may lead to resolvement. The E2-C1 regionmay therefore be particularly useful in therapeutic interventions. Itshould also be noted that, once reactivity to a given domain isestablished, it can be further mapped to smaller peptides, e.g.reactivities of 1 chimpanzee serum to C3 could be mapped to smallerregion of 25 amino acids (peptide C3″).

Example 3 Monitoring of E1 and E2 Antibodies in Patients with Responseto Interferon-Alpha Therapy

In Table 5, results of E2 antibody tests as described in example 2 aregiven for consecutive samples obtained from patients with response tointerferon therapy. A decline in E2Ab, and to a larger extend E1Ab, hasbeen described in PCT/EP 95/03031 in case of a long-term response tointerferon treatment. Reactivities to several peptides of the presentinvention also show similar declining levels. Peculiar reactivitiescould sometimes be detected as exemplified in patient 2: upon thedetection of reappearing virus, antibody responses to the (E1)V4V5region and the (E2)HVRII region could be detected; these quicklydisappeared simultaneously with viral clearance. (E1)V4V5 and (E2)HVRIImay therefore be particularly useful peptides for disease monitoring,especially in treatment of disease. Other peptides such as (E2)C1(example 2) and those shown in bold in Table 5 also seem to be usefulfor purposes such as monitoring. Table 2 also shows the presence ofreactivity in patient 2 to a new peptide HVRI-C1, which overlaps thejunction between HVRI and C1 (Table 2), in the absence of detectablereactivity to the HVRI or C1 peptides. Similarly, peptide C4-bcencompassing the region between C4-b and C4-c (Table 2), was tested inthis series, and showed almost identical reactivities as compared topeptide C4-b. Therefore, it is possible that the C4-b epitope liesbetween aa 658 and 673, but surprisingly, the epitope does not seem tobe presented in peptide SEQ ED 92 of PCT/EP 95/03031 (aa 655-674). TheC4-c epitope is not present in C4-bc and therefore can be localizedbetween aa 683 and 706.

Example 4 Application to Other Flaviviruses

To examine the applicability of the invention to envelope proteins ofother HCV-related viruses, a peptide spanning the V1V2 region of thehepatitis G virus (GBV-C; Linnen et al., 1996; Simons et al., 1996) E1region was synthesized, see also SEQ ID NO 38 (Table 1):NH2-THACRANGQYFLTNCCAPEDIGFCLEGGCLVALGGK-biotin.

So far, only reactivity to the complete HGV E2 protein seemed to beuseful in the diagnosis of HGV. Peptide epitopes have not yet beendescribed for GBV envelope proteins E1 or E2. Sixteen HGV RNA-positivesera were tested and 1 of these was reactive with the E1 peptide asshown in Table 6. Antibody reactivity to the recombinant HGV E2 protein(but not to HGV E2 peptides) is found in up to 15% of the Europeanpopulation, but cases with both HGV RNA and E2Ab are rare as theyprobably represent cases in which seroconversion and elimination of thevirus is ongoing. Antibody reactivity to the HGV E1 protein has not yetbeen reported. Therefore, the HGV E1 peptide V1V2 is new and it maydisplay higher reactivities in a series of HGV anti-E2 reactive sera.Using similar approaches as described in the present invention, HGV E2peptides may also be synthesized. Multimer peptides from GBV-A or GBV-Bcan be synthesized in a similar approach as described for HCV and HGV.

Example 5 Reactivity of 20-Mer E2 Peptides Compared to Multimer E2Peptides

E2 peptides listed in Table 1 were analyzed for their reactivity with 32serum samples from patients with chronic active hepatitis C. Inaddition, a series of overlapping 20-mer peptides were synthesized withexactly the same HCV subtype 1b sequence as used for the longer peptidesand as shown in Table 1. The ELISA test used was the same as describedin Example 2. FIGS. 3 and 4 show the reactivities of the series of20-mer and longer peptides, respectively. Peptides with a sum of >5 (HVRI, HVR I/C1, C1a, C1b, C4a, C4b, C4c, C4b-c) were considered to be veryuseful for the detection of antibodies directed against E2. A total ofsix of these peptides (peptides C4b-c and C1a were not included as thesepeptides are almost entirely represented by other peptides) werecombined together with 20-mer peptide 1350 (Table 1), which occasionallyreacted with some patient sera. The combination of these peptides wastested on a panel of 128 sera from chronic active HCV carriers. Hundredand twenty six of these sera tested positive on recombinant E2s protein.Of these 126 sera, 33 sera showed at least two times higher OD valueswith the peptide mixture as compared to the recombinant E2 protein, 64sera showed a similar reactivity, 16 sera showed reactivities which were2- to 4-fold higher with the recombinant protein than with the peptidemixture, and 13 sera only reacted with the recombinant protein. Insummary, almost 90% of the sera containing antibodies againstrecombinant E2 protein could be detected using the above peptidemixture. For 26% of the sera, detection was even better using thepeptides of the invention, than using recombinant E2 protein. A sum ofOD values of >5, ie exhibited by peptides HVR I, HVR I/C1, C1a, C1b,C4a, C4b, C4c, and C4b-c FIG. 4) is therefore considered a surprisinglyhigh value for the serodiagnosis of antibodies directed against the E2protein of HCV. From the experiment described above, it is also clearthat a combination of recombinant E2 with the peptides of the inventionis a particularly useful composition. Given the variability of the E2protein in different HCV genotypes, the addition of genotype-specificpeptides to recombinant E2 proteins may be a desired way of improvingsensitivity of E2 antibody assays. For example, a variant of peptide C1abased on a reported HCV type 2a sequence HC-J6 could beLINTNGSWHINRTALNCNDSLHTGFLASLFYTHSF, and similar useful variants e.g.based on a genotype 3a sequence, could be synthesized and tested forreactivity. It should be noted that the HCV E2 protein may containinsertions or deletions in any given HCV genotype. For example, whilesubtype 1a and 1b sequences show contiguous sequences which can bealigned without having to insert gaps, HCV type 2a isolates encode E2proteins which are 4 aa's longer as compared to type 1 sequences. Forexample, 2 additional amino acids are inserted in HCV type 2a and 2bsequences around hypervariable region II (HVR II). Therefore, apotentially useful variant of peptide HVRII, based on the HC-J6prototype 2a sequence, would be RSIEAFRVGWGALQYEDNVTNPEDMRPYCW, which isa 30-mer peptide while the subtype 1b sequence-based peptide depicted inTable 1 (SEQ ID 20) is only 28 aa's long. The two glutamates (symbol E)which are inserted in the subtype 2a sequence are shown underlined.Similar peptides can be easily constructed based on sequences andalignments previously published (e.g. Maertens and Stuyver, 1997).

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Yokosuka O, Ito Y, Imazeki F, Ohto M, Omata M. Detection of antibody tohepatitis C E2/NS1 protein in patients with type C hepatitis. BiochBiophys Res Commun 1992; 189: 565-71. TABLE 1 GENO SEQ ID PROTEIN TYPEPEPTIDE AMINO ACID SEQUENCE POSITION NUMBER E1 1a V1V2T1aYQVRNSTGLYHVTNDCPNSSIVYEAADAILHTPGC 192-226 Seq ID 1 1b V1V2T1bYEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGC 192-226 Seq ID 2 2c V1V2T2CVEVKNNSNSYMATNDCSNSSIIWQLEGAVLHTPGC 192-226 Seq ID 3 2c V1V2T2cVEVKNTSTSYMVTNDCSNSSIVWQLEGAVLHTPGC 192-226 Seq ID 4 3a V1V2T3aLEWRNTSGLYVLTNDCSNSSIVYEADDVILHTPGC 192-226 Seq ID 5 3a V2T3aLTNDCSNSSIVYEADDVILHTPGC 203-226 Seq ID 6 4c/4k V1V2T4aINYRNVSGIYHVTNDCPNSSIVYEADHHILHLPGC 192-226 Seq ID 7 5a V1V2T5aVPYRNASGIYHITNDCPNSSIVYEADNLILHAPGC 192-226 Seq ID 8 6a V1V2T6aLTYGNSSGLYHLTNDCSNSSIVLEADAMILHLPGC 192-226 Seq ID 9 1b V2V3IVYEAADMIMHTPGCVPCVRENNSSRCWV 212-240 Seq ID 10 1b V3V4VRENNSSRCWVALTPTLAARNASVPTTTIRRHVD 230-263 Seq ID 11 1b PC-V3V4PCVRENNSSRCWVALTPTLAARNASVPTTTIRRHVD 228-263 Seq ID 12 1b HRHVDLLVGAAAFCSAMYVGDLCGSVFLVSQL 260-290 Seq ID 13 1b V5C4SQLFTISPRRHETVQDCNCSIYPGHITGHRMAWDMMMNWS 288-327 Seq ID 14 1b C4V6SIYPGHITGHRMAWDMMMNWSPTTALWSQLLRI 307-340 Seq ID 15 1b SAPQAVVDMVAGAHWGVLAGLAYYSMVGNWAKVLVVMLLFAGV 341-381 Seq ID 16 1b VAV5VALTPTLAARNASVPTTTIRRHVDSQLFTISPRRHETVQD 240-303 Seq ID 37 E1(HGV) NDV1V2 THACRANGQYFLTNCCAPEDIGFCLEGGCLVALGGK ND Seq ID 38 E2 1b HVR IHTRVSGGAAASNTRGLVSLFSPGSAQKIQLVN 384-415 Seq ID 17 1b C1aLVNTNGSWHINRTALNCNDSLQTGFFAALFYKHKF 413-447 Seq ID 18 1b C1bNDSLQTGFFAALFYKHKFNSSGCPERLASCRSIDKFAQ 430-467 Seq ID 19 1b HVR IIRSIDKFAQGWGPLTYTEPNSSDQRPYCW 460-487 Seq ID 20 1b C2aSDQRPYCWHYAPRPCGIVPASQVCGPVYCFTPSP 480-513 Seq ID 21 1b C2bSQVCGPVYCFTPSPVVVGTTDRFGVPTYNWG 500-530 Seq ID 22 1b V3C3GVPTYNWGANDSDVLILNNTRPPRGNWFGCTWMNGTGFTKTCGG 523-566 Seq ID 23 1b V3C3′ANDSDVLILNNTRPPRGNWFGCTWMNGTGFTKTCGG 531-566 Seq ID 24 1b C3″TRPPRGNWFGCTWMNGTGFTKTCGG 542-566 Seq ID 25 1b V4TKTCGGPPCNIGGAGNNTLTCPTDCFRKHP 561-590 Seq ID 26 1b C4TDCFRKHPEATYARCGSGPWLTPRCMVHYPYRLWHYPCTVNFTIF 583-627 Seq ID 27 1b C4′ARCGSGPWLTPRCMVHYPYRLWHYPCTVNFTIF 595-627 Seq ID 28 1b C4″LTPRCMVHYPYRLWHYPCTVNFTIF 603-627 Seq ID 29 1b C4aTVNFTIFKVRMYVGGVEHRFEAACNWTR 621-648 Seq ID 30 1b C4bEAACNWTRGERCDLEDRDRSELSPLLLSTTEWQ 641-673 Seq ID 31 1b C4cQWQILPCSFTTLPALSTGLIHLHQNIVDVQYLYGVG 671-706 Seq ID 32 E2 1b SAGVGSAVVSLVIKWEYVLLLFLLLADARICACLWMMLLIAQAE 704-745 Seq ID 33 1b HVR I/C1NTRGLVSLFSPGSAQKIQLVNTNGSWHINRTALN 395-428 Seq ID 34 1b CAb-cDRSELSPLLLSTTEWQILPCSFTTLPALSTG 658-688 Seq ID 35 1b 1350VGTTDRFGVPTYNWGANDSD 516-535 Seq ID 36

TABLE 2 HVR Rec Sample # HVR I C1-a C1-b II C2-a C2-b E2-13 B C3 C3′ C3″V4 C4 C4-a C4-b C4-c SA E2 17758 69 48 47 52 49 48 47 49 38 44 43 52 4455 48 46 1355 17763 88 54 44 49 52 48 51 51 46 45 48 49 45 133 104 50361 17764 100 148 138 134 128 136 141 136 136 65 130 145 144 242 128 127371 17766 91 97 145 96 80 87 90 90 95 47 75 89 163 139 99 86 173 17771307 79 54 65 51 50 65 68 50 45 60 65 59 96 132 58 393 17775 49 50 46 3950 271 43 51 48 45 50 55 52 54 47 50 228 17777 60 133 105 130 129 123118 118 130 95 119 133 129 357 177 113 850 17779 373 328 285 330 284 343281 323 316 283 297 318 343 341 309 282 720 17785 81 80 73 71 76 66 8170 74 70 69 79 79 87 119 73 146 17786 341 863 693 152 164 179 148 139146 136 137 158 160 163 148 157 720 17788 111 553 120 137 69 121 121 119111 110 103 140 132 131 48 47 934 17789 1316 49 47 46 49 45 53 51 48 4342 50 49 52 48 48 1178 17790 234 233 182 223 130 224 185 185 186 184 179216 218 1347 853 207 1534 17791 269 194 177 192 123 203 172 192 157 184184 200 195 211 187 190 287 17797 260 264 248 257 240 281 249 237 246221 223 283 261 272 231 243 1357 17798 52 53 50 47 52 54 50 53 49 51 5051 50 1036 51 51 1161 17799 225 89 81 86 85 100 76 85 87 82 84 86 92 11586 76 362 17802 42 51 44 47 50 133 48 52 51 48 51 56 76 773 157 56 88217807 49 133 60 59 66 62 62 59 57 56 57 63 65 62 57 52 605 17808 89 121117 109 106 1051 118 875 133 116 123 126 393 228 109 126 1354 17810 327220 199 222 195 200 221 182 197 182 196 209 266 222 195 199 422 17818224 134 115 126 118 115 128 108 109 98 111 113 112 117 109 108 230 17821671 243 214 282 238 232 228 217 234 197 216 222 218 557 810 205 104617825 397 320 264 284 282 286 289 277 276 274 276 306 273 391 399 277514 17826 92 109 111 99 114 126 113 98 104 84 105 121 122 126 145 113695 17827 45 47 46 47 48 49 48 49 49 47 49 50 50 261 113 47 320 17832151 65 55 70 78 63 77 72 68 59 64 70 62 54 57 49 288 17838 212 167 166164 156 165 164 146 160 154 150 165 165 161 272 157 305 17839 48 94 11761 61 51 58 51 46 52 58 55 87 60 95 66 182 17840 318 323 347 317 329 338320 305 326 302 312 343 355 322 318 337 417 17842 161 174 185 176 168163 159 157 163 156 150 168 151 154 138 153 195 17844 122 94 90 88 98 7892 88 84 77 85 94 61 214 51 73 166 17849 1469 68 75 49 54 629 52 53 4646 51 54 119 1102 55 47 1393 17870 125 236 148 114 128 133 135 116 132109 135 151 118 293 120 45 197 17879 209 195 201 222 195 215 225 191 194181 218 209 209 255 253 199 325 17983 438 54 50 48 52 46 50 54 46 46 5152 46 55 53 48 216 17999 276 201 200 202 190 187 191 169 176 150 190 205186 321 535 198 697  8242 162 114 114 127 140 114 120 117 103 120 117107 112 161 152 128 340  8243 188 191 171 175 204 172 189 174 186 174176 205 200 206 177 178 225  8247 248 169 137 127 120 110 122 96 111 104114 128 104 130 150 118 215  8250 129 161 127 150 164 144 154 125 134122 142 151 125 146 137 140 165  8317 112 131 115 123 113 111 144 95 10395 108 118 108 158 126 111 198  8320 463 433 337 473 435 445 363 345 503384 362 369 405 446 432 378 474  8329 119 126 123 160 143 145 142 117135 121 122 126 131 152 148 132 163  8330 198 271 210 210 207 196 216178 194 206 209 215 186 356 45 51 536  8332 154 141 128 141 132 116 129110 123 112 135 140 123 147 312 144 290  8333 57 67 50 51 52 52 50 54 5050 50 56 48 480 65 52 1108  8334 283 66 64 80 68 69 84 79 65 52 67 74 72180 191 90 348  8337 162 105 99 108 103 92 104 86 93 80 101 107 108 124118 110 142  8339 50 49 52 62 54 46 54 51 47 41 51 55 53 413 49 50 247 8344 59 52 50 51 58 48 54 52 47 48 55 53 58 63 63 60 59  8351 163 114105 111 101 91 98 97 92 78 110 111 115 141 179 112 154  8362 211 54 5047 55 119 53 53 44 45 51 54 59 60 58 55 165  8364 110 308 106 112 112107 98 102 108 92 116 152 133 208 169 132 671  8365 69 84 94 67 77 74 5573 70 69 70 79 73 69 88 66 86  8367 218 189 171 201 204 174 191 156 158140 183 186 294 197 186 171 303  8374 575 113 95 114 110 93 100 92 10688 103 125 118 112 111 106 143  8377 364 232 229 225 211 202 233 189 207170 209 205 230 234 218 221 293  8382 314 211 187 196 207 173 208 181158 150 181 187 201 223 189 211 265  8383 51 100 102 55 58 48 57 53 5350 52 57 66 94 63 56 285 V1200 52 55 52 56 55 53 50 54 50 52 51 50 50 5253 54 50 V1201 118 147 138 136 224 144 123 137 140 111 135 154 166 171137 155 162 V1202 274 308 284 170 290 286 282 248 277 229 271 306 287330 268 295 329 V1204 130 134 135 127 141 128 79 113 119 106 131 144 145144 130 144 159

TABLE 3 E1 antigens Sample# No peptide V1V2 V2V3 V3V4 HR/SA V5 C4V6 recE1s No sample 0.011 0.007 0.011 0.014 0.009 0.007 0.009 0.056 30108 0.030.035 0.04 0.034 0.032 0.03 0.234 0.378 30109 0.032 0.033 0.035 0.0280.024 0.026 0.227 0.368 30110 0.021 0.545 0.02 0.019 0.016 0.017 0.0470.669 30111 0.017 0.614 0.019 0.018 0.017 0.015 0.064 0.796 30112 0.0370.069 0.035 0.034 0.031 0.031 0.048 0.187 30113 0.042 0.083 0.136 0.0390.034 0.035 0.063 0.226 30114 0.042 0.099 0.036 0.035 0.035 0.037 0.0580.267 30115 0.021 0.114 0.023 0.021 0.02 0.02 0.189 0.339 30116 0.0190.442 0.025 0.022 0.022 0.018 0.056 0.645 30117 0.027 0.062 0.047 0.0430.041 0.038 0.066 0.164 30118 0.122 0.216 0.126 0.12 0.11 0.125 0.6960.923 30119 0.023 0.028 0.031 0.028 0.023 0.024 0.23 0.426 30120 0.0250.024 0.027 0.025 0.039 0.027 0.03 0.024 30121 0.03 0.033 0.033 0.0290.052 0.034 0.037 0.032 30122 0.029 0.031 0.056 0.03 0.052 0.033 0.0350.03 30123 0.085 0.081 0.076 0.075 0.087 0.071 0.094 0.137 30124 0.0220.084 0.022 0.022 0.023 0.022 0.193 0.391 30125 0.095 0.128 0.091 0.0890.172 0.159 0.47 0.708 17805 0.038 0.051 0.039 0.033 0.09 0.154 0.7381.169 13059 0.011 0.011 0.012 0.012 0.014 0.012 0.229 0.681 Chimp1 0.0950.38 0.276 0.126 0.098 0.095 0.099 0.805 Chimp2 0.026 0.234 0.143 0.0350.036 0.038 0.354 0.822 Chimp3 0.018 0.017 0.02 0.022 0.023 0.019 0.1410.353

TABLE 4 E2 antigens Sample peptide HVR I C1-a C1-b HVR II C2-a C2-b C3C3′ C3″ V4 C4 C4-a C4-b C4-c recE2h No sample 0.006 0.009 0.011 0.0150.007 0.006 0.01 0.01 0.01 0.01 0.01 0.01 0.007 0.007 0.009 0.032 301080.036 0.747 0.848 0.969 0.032 0.033 0.03 0.04 0.02 0.02 0.03 0.03 0.0410.026 0.031 0.988 30109 0.027 0.849 0.93 1.053 0.027 0.032 0.03 0.030.02 0.02 0.03 0.02 0.038 0.023 0.026 1.079 30110 0.018 0.026 0.0210.044 0.019 0.024 0.02 0.02 0.02 0.02 0.02 0.03 0.023 0.026 0.056 0.1130111 0.017 0.02 0.021 0.088 0.018 0.02 0.02 0.02 0.01 0.02 0.02 0.030.022 0.028 0.07 0.137 30112 0.037 0.092 0.052 0.177 0.044 0.048 0.040.04 0.04 0.04 0.04 0.05 0.043 0.562 0.053 0.947 30113 0.045 0.104 0.0540.276 0.051 0.047 0.05 0.03 0.04 0.04 0.04 0.05 0.054 0.633 0.07 1.00330114 0.045 0.112 0.075 0.726 0.046 0.041 0.05 0.05 0.03 0.04 0.04 0.060.054 0.646 0.067 1.065 30115 0.022 0.982 0.034 0.064 0.025 0.025 0.020.03 0.02 0.03 0.03 0.02 0.03 0.097 0.031 0.413 30116 0.015 0.023 0.020.04 0.017 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.023 0.022 0.046 0.08430117 0.04 0.087 0.048 0.119 0.037 0.044 0.05 0.05 0.03 0.04 0.04 0.040.041 0.547 0.049 0.935 30118 0.112 0.213 0.122 0.119 0.119 0.121 0.120.12 0.11 0.05 0.11 0.1 0.117 0.105 0.2 0.289 30119 0.03 0.954 1.0121.128 0.026 0.029 0.03 0.03 0.02 0.02 0.03 0.03 0.035 0.026 0.03 1.12330120 0.031 0.427 0.208 0.208 0.03 0.033 0.03 0.04 0.03 0.03 0.03 0.030.033 0.032 0.032 0.577 30121 0.033 0.734 0.463 0.398 0.037 0.042 0.040.05 0.04 0.03 0.04 0.03 0.04 0.034 0.037 0.963 30122 0.03 0.661 0.4130.365 0.043 0.034 0.03 0.04 0.04 0.03 0.03 0.03 0.038 0.03 0.034 0.90730123 0.079 0.11 0.576 0.789 0.09 0.108 0.09 0.08 0.08 0.06 0.07 0.060.091 0.078 0.077 0.916 30124 0.02 0.939 0.041 0.065 0.028 0.237 0.040.04 0.02 0.03 0.02 0.02 0.038 0.108 0.049 0.4 30125 0.096 0.133 0.1030.096 0.097 0.115 0.15 0.14 0.09 0.09 0.09 0.1 0.1 0.092 0.183 0.22717805 0.042 0.255 0.074 0.078 0.071 0.045 0.06 0.06 0.05 0.04 0.06 0.040.163 0.043 0.831 0.881 13059 0.013 0.47 0.02 0.019 0.018 0.022 0.020.03 0.02 0.01 0.01 0.02 0.36 0.052 0.904 0.944 Chimp1 0.102 0.103 0.1160.118 0.23 0.109 0.12 0.19 0.17 0.19 0.1 0.1 0.087 0.098 0.095 0.581Chimp2 0.028 0.181 0.267 0.261 0.056 0.032 0.03 0.04 0.04 0.04 0.04 0.040.188 0.035 0.033 1.008 Chimp3 0.058 0.035 0.162 0.086 0.026 0.062 0.020.03 0.04 0.02 0.03 0.03 0.023 0.02 0.026 1.327

TABLE 5 HCV E1 peptides Sample PCR Genotype V1V2 V2V3 V3V4 V4V5 HR/SAV5C4 C4V6 E1s Patient 1 14/8/90 pos 3a 0.014 0.03 0.06 0.034 0.037 0.0480.045 0.051 01/06/91 0.03 0.032 0.064 0.041 0.041 0.051 0.048 0.04520/9/91 neg 0.06 0.064 0.064 0.037 0.039 0.05 0.398 0.045 13/3/92 0.0340.041 0.037 0.034 0.037 0.046 0.044 0.04 04/09/92 neg 0.037 0.041 0.0390.037 0.037 0.052 0.048 0.043 24/9/93 0.048 0.051 0.05 0.046 0.052 0.0480.047 0.042 20/10/94 neg 0.045 0.048 0.398 0.044 0.048 0.047 0.045 0.04123/10/95 0.051 0.045 0.045 0.04 0.043 0.042 0.041 0.051 10/12/96 pos?0.037 0.041 0.033 0.034 0.035 0.039 0.038 0.045 Patient 2 15/2/90 0.1060.103 0.104 0.108 0.104 0.949 0.872 1.03 03/05/90 pos 1a 0.103 0.1090.106 0.104 0.108 0.828 0.859 1.04 04/12/90 0.096 0.103 0.105 0.1030.095 0.737 0.848 1.218 23/9/91 0.063 0.078 0.078 0.067 0.072 0.3180.354 0.66 14/4/92 0.099 0.106 0.099 0.1 0.096 0.219 0.255 0.49118/12/92 0.104 0.106 0.102 0.105 0.101 0.222 0.249 0.448 26/3/93 0.0890.095 0.09 0.085 0.082 0.168 0.194 0.357 30/9/93 neg 0.092 0.081 0.0890.09 0.088 0.17 0.18 0.35 17/6/94 pos 1a 0.084 0.09 0.096 0.599 0.0950.154 0.166 0.32 18/12/95 0.072 0.077 0.077 0.077 0.081 0.111 0.1210.206 23/12/96 neg 0.065 0.078 0.074 0.073 0.078 0.106 0.108 0.199Patient 3 15/04/93 0.005 0.006 0.005 0.004 0.006 0.005 0.006 0.00706/09/94 pos 3a 0.007 0.008 0.007 0.008 0.007 0.006 0.006 0.009 30/10/95neg 0.007 0.01 0.009 0.009 0.009 0.008 0.007 0.011 18/11/96 pos? 1b0.012 0.012 0.012 0.011 0.01 0.009 0.009 0.012 Patient 4 12/04/91 pos 1a0.006 0.007 0.006 0.006 0.007 0.006 0.006 0.01 23/09/91 neg 0.01 0.010.008 0.009 0.009 0.006 0.008 0.013 27/07/92 neg 0.007 0.009 0.007 0.0080.007 0.006 0.007 0.01 11/06/93 neg 0.009 0.011 0.009 0.01 0.009 0.0070.006 0.011 29/11/96 pos 1a 0.007 0.01 0.008 0.007 0.007 0.005 0.0060.008 Patient 5 18/09/92 pos 0.017 0.01 0.008 0.007 0.008 0.178 0.1960.537 17/12/93 neg 0.012 0.014 0.011 0.01 0.011 0.039 0.04 0.23115/11/96 neg 0.012 0.014 0.012 0.01 0.01 0.026 0.017 0.116 Patient 610/05/90 pos 0.311 0.006 0.007 0.005 0.006 0.004 0.01 0.544 11/10/91 neg0.284 0.007 0.007 0.006 0.007 0.006 0.013 0.605 Patient 7 10/10/91 pos1b 0.009 0.01 0.009 0.008 0.008 0.008 0.01 0.043 18/12/92 neg 0.01 0.0110.011 0.009 0.009 0.008 0.011 0.043 28/06/93 neg 0.006 0.006 0.007 0.0060.007 0.005 0.008 0.021 10/03/97 pos 1b 0.008 0.008 0.007 0.008 0.0070.006 0.008 0.012 Patient 8 19/08/91 neg 0.008 0.009 0.008 0.008 0.0080.006 0.008 0.009 17/07/95 pos 1b 0.01 0.009 0.009 0.009 0.006 0.0070.007 0.018 09/10/95 pos 1b 0.007 0.007 0.008 0.005 0.006 0.007 0.0070.009 15/12/95 neg 0.008 0.009 0.008 0.009 0.008 0.007 0.007 0.01104/03/96 neg 0.009 0.011 0.01 0.011 0.009 0.008 0.007 0.01 02/09/96 neg0.01 0.011 0.011 0.01 0.01 0.008 0.008 0.013 Patient 9 26/08/91 pos1b/2ac 0.044 0.015 0.022 0.023 0.028 0.031 0.034 0.115 21/12/93 neg0.033 0.017 0.021 0.027 0.022 0.025 0.023 0.048 20/12/94 pos 1b 0.0230.016 0.015 0.028 0.019 0.028 0.034 0.077 21/12/95 pos 1b 0.019 0.0290.024 0.027 0.027 0.031 0.034 0.048 Patient 10 27/04/92 pos 1b 0.1280.024 0.02 0.023 0.026 0.118 0.449 0.68 01/06/93 neg 0.107 0.03 0.0290.027 0.026 0.098 0.385 0.667 Patient 11 09/11/90 neg 0.018 0.019 0.0120.013 0.015 0.087 0.141 0.591 12/07/91 pos 1b 0.023 0.023 0.016 0.020.018 0.073 0.1 0.466 28/05/93 pos 0.008 0.009 0.009 0.005 0.008 0.1230.173 0.495 20/01/95 neg 0.011 0.009 0.008 0.007 0.007 0.026 0.047 0.18708/01/96 neg 0.012 0.013 0.01 0.009 0.009 0.025 0.031 0.21 07/02/97 neg0.019 0.019 0.014 0.014 0.013 0.027 0.051 0.203 Patient 12 11/05/92 pos1b 0.017 0.013 0.011 0.014 0.015 0.227 0.173 0.425 26/02/93 neg 0.0220.014 0.013 0.013 0.014 0.178 0.264 0.417 12/08/93 pos 1b 0.016 0.0160.016 0.014 0.015 0.29 0.387 0.63 Patient 13 07/01/91 pos 1b 0.027 0.0170.021 0.026 0.026 0.04 0.074 0.062 19/08/91 neg 0.018 0.018 0.015 0.0130.012 0.021 0.009 0.043 21/08/92 pos 0.015 0.012 0.015 0.014 0.017 0.0150.021 0.023 06/08/93 neg 0.019 0.018 0.016 0.021 0.016 0.01 0.011 0.0206/03/95 pos 1b 0.027 0.026 0.018 0.015 0.018 0.02 0.023 0.028 12/04/96neg 0.03 0.017 0.018 0.036 0.021 0.027 0.027 0.022 Patient 14 22/11/94pos 1b 0.016 0.011 0.013 0.013 0.026 0.318 0.437 0.461 11/10/95 pos0.024 0.014 0.014 0.018 0.019 0.039 0.061 0.059 15/02/96 neg 0.032 0.0220.021 0.023 0.016 0.031 0.041 0.102 Patient 15 04/12/90 pos 1b 0.0030.005 0.005 0.004 0.005 0.005 0.005 0.019 29/11/90 neg 0.005 0.005 0.0050.006 0.005 0.008 0.006 0.011 09/10/92 pos 1b 0.006 0.008 0.007 0.0070.007 0.006 0.005 0.012 25/03/96 neg 0.006 0.008 0.007 0.006 0.006 0.0040.007 0.012 Patient 16 16/12/91 pos 3a 0.003 0.004 0.006 0.004 0.0040.08 0.102 0.435 04/10/93 neg 0.006 0.007 0.007 0.006 0.008 0.028 0.0330.253 12/09/94 neg 0.004 0.008 0.006 0.005 0.005 0.034 0.038 0.19709/09/96 neg 0.004 0.008 0.007 0.006 0.005 0.008 0.013 0.08 Patient 1724/04/97 pos 1b 0.076 0.006 0.008 0.004 0.009 0.203 0.327 1.196 Patient18 08/01/97 neg 0.006 0.007 0.007 0.007 0.006 0.006 0.008 0.009 Blank0.006 0.009 0.009 0.006 0.006 0.007 0.006 0.009

TABLE 6 Sample# Blank E1 V1V2 20188 68 74 20189 77 73 20251 170 15020252 490

20253 92 70 20254 50 55 20255 81 88 20256 56 62 20266 119 134 20271 7778 20272 61 69 21010 129 135 21011 159 161 21012 120 93 21286 108 105

1-22. (canceled)
 23. An isolated HCV E1 envelope peptide as defined byany of SEQ ID Nos:1-16 and
 37. 24. An isolated HCV E1 envelope peptideconsisting of up to 45 contiguous amino acids wherein an amino acidsequence selected from SEQ ID Nos:1-16 and 37 is present in saidpeptide.
 25. An isolated peptide selected from the group consisting of:a peptide of 21 to 23 contiguous amino acids of SEQ ID NO:6; a peptideof 21 to 28 contiguous amino acids of SEQ ID NO:10; a peptide of 21 to30 contiguous amino acids of SEQ ID NO:13; a peptide of 21 to 33contiguous amino acids of SEQ ID NO:11 or 15; a peptide of 21 to 34contiguous amino acids of SEQ ID NOs:1-5 or 7-9; a peptide of 21 to 35contiguous amino acids of SEQ ID NO:12; a peptide of 21 to 39 contiguousamino acids of SEQ ID NO:14 or 37; a peptide of 21 to 40 contiguousamino acids of SEQ ID NO:16.
 26. A method of immunizing a human againstinfection with HCV-related virus or any mutated strain thereof,comprising administering to said human at least one peptide according toclaim
 23. 27. An assay kit for detecting the presence ofanti-HCV-related virus antibodies within a sample of body fluidcomprising: optionally, a solid support, at least one peptide accordingto claim 23, and optionally, markers which allow detection of complexesformed between anti-HCV-related virus antibodies within a sample of bodyfluid with said at least one peptide.
 28. A bioassay for identifying acompounds which modulate the interaction between a peptide according toclaim 23 and an anti-HCV-related virus antibody, said bioassaycomprising (i) contacting said peptide with said anti-HCV-related virusantibody; (ii) after (i), determining the binding between said peptideand said anti-HCV-related virus antibody; (iii) adding said compound ora combination of said compounds to the peptide-antibody complex formedin (i); (iv) after (iii), determining the binding between said peptideand said anti-HCV-related virus antibody; and (v) inferring, from (ii)and (iv) the modulation of binding between said peptide and saidanti-HCV-related virus antibody by said added compound or said addedcombination of compounds.
 29. A bioassay for identifying a compoundswhich modulate the interaction between a peptide according to claim 23and an anti-HCV-related virus antibody, said bioassay comprising (i)determining the binding between said peptide and said anti-HCV-relatedvirus antibody; (ii) contacting said peptide with said compound; (iii)adding said anti-HCV-related virus antibody to the peptide-compoundcomplex formed in (ii); (iv) after (iii), determining the bindingbetween said peptide and said compound; (v) inferring, from (i) and (iv)the modulation of binding between said peptide and said anti-HCV-relatedvirus antibody by said compound.
 30. The isolated peptide of claim 23which is synthesized chemically.
 31. The isolated peptide of claim 23which is synthesized using recombinant DNA techniques.
 32. The isolatedpeptide of claim 31 wherein said peptide is synthesized using a plasmidvector comprising a nucleotide sequence encoding said peptide operablylinked to transcription regulatory elements.
 33. The isolated peptide ofclaim 23 which is biotinylated or which is containing cysteine bridges.34. The isolated peptide of claim 23 which binds and recognizes anti-HCVvirus antibodies.
 35. The isolated peptide of claim 33 which binds andrecognizes anti-HCV virus antibodies.
 36. A combination of peptidescomprising a peptide of claim
 23. 37. A combination of peptidescomprising a peptide of claim
 33. 38. A combination of peptidescomprising a peptide of claim
 34. 39. A composition comprising anisolated peptide of claim
 23. 40. A composition comprising an isolatedpeptide of claim
 33. 41. A composition comprising an isolated peptide ofclaim
 34. 42. An assay kit for detecting the presence of anti-HCV virusantibodies within a sample of body fluid comprising at least one peptideof claim
 23. 43. An assay kit for detecting the presence of anti-HCVvirus antibodies within a sample of body fluid comprising a combinationof peptides of claim
 33. 44. An assay kit for detecting the presence ofanti-HCV virus antibodies within a sample of body fluid comprising acombination of peptides of claim
 34. 45. An assay kit for detecting thepresence of anti-HCV virus antibodies within a sample of body fluidcomprising a combination of peptides of claim
 36. 46. An assay kit fordetecting the presence of anti-HCV virus antibodies within a sample ofbody fluid comprising a combination of peptides of claim
 37. 47. Amethod of raising an immune response in a human against hepatitis Cvirus, comprising administering to said human at least one peptideaccording to claim
 23. 48. A method of raising an immune response in ahuman against hepatitis C virus, comprising administering to said humanat least one peptide according to claim
 30. 49. A method of raising animmune response in a human against hepatitis C virus, comprisingadministering to said human at least one peptide according to claim 31.50. A method of raising an immune response in a human against hepatitisC virus, comprising administering to said human at least one peptideaccording to claim
 32. 51. A method of raising an immune response in ahuman against hepatitis C virus, comprising administering to said humanat least one peptide according to claim
 33. 52. A method of raising animmune response in a human against hepatitis C virus, comprisingadministering to said human a combination of peptides according to claim23.
 53. A method for diagnosing exposure to or infection by HCV virusescomprising: contacting anti-HCV virus antibodies within a sample of bodyfluid with at least one peptide according to claim 23, determining thebinding of anti-HCV virus antibodies within a sample of body fluid withsaid at least one peptide.
 54. The method according to claim 53 whereinsaid anti-HCV virus antibodies are anti-HCV antibodies.
 55. An assay kitfor detecting the presence of anti-HCV antibodies within a sample ofbody fluid comprising: optionally, a solid support, at least one peptideaccording to claim 23, and optionally, markers which allow detection ofcomplexes formed between anti-HCV antibodies within a sample of bodyfluid with said at least one peptide.
 56. A bioassay for identifying acompounds which modulate the interaction between a peptide according toclaim 23 and an anti-HCV antibody, said bioassay comprising (i)contacting said peptide with said anti-HCV antibody; (ii) after (i),determining the binding between said peptide and said anti-HCV antibody;(iii) adding said compound or a combination of said compounds to thepeptide-antibody complex formed in (i); (iv) after (iii), determiningthe binding between said peptide and said anti-HCV antibody; and (v)inferring, from (ii) and (iv) the modulation of binding between saidpeptide and said anti-HCV antibody by said added compound or said addedcombination of compounds.
 57. A bioassay for identifying a compoundswhich modulate the interaction between a peptide according to claim 23and an anti-HCV antibody, said bioassay comprising (i) determining thebinding between said peptide and said anti-HCV antibody; (ii) contactingsaid peptide with said compound; (iii) adding said anti-HCV-relatedvirus antibody to the peptide-compound complex formed in (ii); (iv)after (iii), determining the binding between said peptide and saidcompound; (v) inferring, from (i) and (iv) the modulation of bindingbetween said peptide and said anti-HCV antibody by said compound.